On the clinical value of genetic analysis in colorectal cancer patients

Sammanfattning: In Sweden, the incidence of colorectal cancer (CRC) is about 5000 new cases every year and this number has not changed markedly during the past twenty years. 90-95% of all CRC appear spontaneously, but there are also inherited forms of CRC (5-10%) of which the two most common are familiar adenomatous polyposis (FAP) and hereditary non-polyposis colorectal carcinoma (HNPCC). A set-up of molecular variables that would enable us to improve tumour staging would help to avoid unnecessary adjuvant treatment and surveillance in many cases. Acquired CRC is thought to occur as a consequence of clonal accumulation of DNA alterations and the accumulation of these growthpromoting events is the basis of the multistep carcinogenesis. Among the most well described loci subject to alterations engaging tumour suppressor genes in CRC are the APC gene (at chromosome 5q), the DCC gene (at chromosome 18q) and the p53 gene (at chromosome 17p). Additionally, point mutations of the k-ras oncogene have been found in about 50% of adenomas and CRC. The results are often conflicting as to whether a certain DNA aberration may be of clinical value as a predictor of outcome. This work focuses on the biological and genetic heterogeneity within the colorectal tumour mass. In paper I, we evaluate the importance of intratumoural heterogeneity throughout the tumour in CRC, and to what extent this assumed heterogeneity may affect the results. We show, that with single biopsy procedures, which are almost exclusively used in these studies, the chance of getting a relevant biopsy is about 50% when analyzing loss of heterozygosity (LOH) at chromosome 17p and 18q. The need for obtaining biopsies under microscope and a multiple biopsy procedure is emphasized. In paper II, we study to what degree intratumoural distribution of genetic aberrations (the status of LOH 5q, 17p, 18q and k-ras mutations) is dependant on histopathological intratumoural subclassification in CRC. This work demonstrates a correlation between intratumoural differences in the degree of neoplastic dedifferentiation and genetic instability in terms of allelic loss and point mutations of the kras gene. Additionally, the occurrence of these genetic alterations within preneoplastic areas as well as in normal colonic mucosa of these patients is described. The genetic lesions studied in this thesis reportedly occur in 35-70 % of colorectal tumours, and many studies have attributed these mutations to have a prognostic significance. In paper III, we challenge these data by dividing colorectal tumours totally into 5x5x5 millimetre "biopsies". From these DNA was extracted and serially analysed until mutations were detected. In the 17 patients studied we found LOH at 5q, 17p, and 18q in 98%. In one of the tumours, LOH at 5q could not be detected. The frequency of LOH-positive biopsies varied widely within the tumors. In contrast, k-ras mutations were found to occur practically in all biopsies throughout the tumours in 7/17 (41%) and not at all in 9/17 (53%). Our results are consistent with the concept that k-ras is an early, but not a necessary event in tumour development. Furthermore, the study clearly demonstrates that allelic loss cannot be used as a prognostic marker in CRC. In Paper IV, we study patients with an ileoanal pelvic pouch after colectomy. Biopsies from patients with chronically inflamed pelvic pouches with known preneoplastic mucosal changes were screened for chromosomal alterations (k-ras, allelic loss) associated with CRC. We found that DNA aberrations are rare in these patients. Nevertheless, dysplasia, aneuploidy and LOH 5q, which were detected in one patient, may reflect different parts of an atrophic mucosa-dysplasia-carcinoma sequence. In Paper V, we explore the use of PCR-based methods for the detection of free circulating cancer specific DNA in plasma and serum. We studied patients undergoing surgery for CRC plasma pre- and postoperatively and compared the tumour status with the presence of genetic alterations. Fully developed, plasma DNA analysis may have a role as a clinical tool in the care of these patients.